Mucin 1 (MUC1) is a highly O-glycosylated protein normally expressed on epithelial, endothelial and other normal cell types. MUC1 and other members of the mucin family provide lubrication of cell surfaces and serve as a barrier to physical and biological assaults on the cell. However, in human tumors mucins are often aberrantly expressed. This is believed to contribute to tumor progression by altering the surface properties of tumor cells. MUC1 is one of the best characterized of the mucins. It has a role in cell adhesion and has been shown to interact with various signaling proteins involved in cellular growth.
MUC1 has been exclusively localized to the apical surfaces of normal epithelial cells of breast, salivary gland and lung. The protein backbone has both highly glycosylated regions and unglycosylated regions. The protein core exhibits of a variable number of tandem repeats (VNTR) each 20 amino acids in length. The carbohydrate side chains are attached by an α-linkage between N-acetylgalactosamine and the oxygen atom of serine or threonine. Each VNTR contains five potential sites for glycosylation.
Humans are generally immunologically tolerant to the human MUC1 protein core and its normal glycoforms. However, MUC1 is up-regulated in many malignant tumors. In addition, cancer-associated MUC1 is underglycosylated (i.e., hypoglycosylated) and exhibits an abnormal number of truncated immature O-glycan side-chains. The changes in the carbohydrate structure of cancer-associate MUC1 distinguish it structurally from MUC1 found in normal tissues. As a consequence of hypoglycosylation, regions of the protein backbone that are normally masked by carbohydrate become exposed, making the cancer-associate molecule antigenically distinct from the MUC1 found in normal epithelia.
Because MUC1 is over-expressed in many cancers, it has been used as a serum marker for detecting recurrence or prognosis in cancer patients, particularly breast cancer patients. The MUC1 glycoforms associated with normal breast epithelial cells are core 2 glycoforms that are characterized by branching at the 6-position of the GalNAc residue. In contrast, in breast cancer core 1 glycoforms predominate. Core 1 glycoforms are characterized by a lack of branching at the 6-position of GalNAc. The failure of breast cancer cells to produce normally branched glycoforms may be due to a decrease in expression of core2β 6-GlcNAc transferase (C2GnT1). Anti-MUC1 antibodies have also been employed as immunohistochemical markers in the diagnosis of such cancers. Anti-MUC1 antibodies directed against different regions of MUC1, including the VNTR, are known.
Cellular immune responses to MUC1 have been extensively characterized in cancer patients. Humoral immune responses to MUC1 in malignancy have also been reported and correlated with improved outcome in breast cancer. There is therefore interest in developing antibodies to MUC1 and immunogens based on the MUC1 polypeptide for use in immunotherapy for patients with cancer for use in treating tumors expressing the MUC1 antigen. It is also of interest to elicit cellular and humoral immune responses directed specifically to cancer-associated glycoforms of MUC1 to improve the therapeutic effect.
The heptapeptide sequence Pro-Asp-Thr-Arg-Pro-Ala-Pro (PDTRPAP, SEQ ID NO:1) is derived from the VNTR of MUC1 and has been identified as the epitope recognized by anti-KL-6 monoclonal antibody (anti-KL-6MAb). Ohyabu, N. J., et al. Am. Chem. Soc. 2009; 131(47):17102-9. The threonine residue of this epitope is modified by Neu5Ac alpha 2,3 Gal beta 1,3 GalNAc alpha (2,3 sialyl T antigen or “23ST”, a core 1-type O-glycan). The authors report that the antibody is not specific for this epitope structure, and that it reacts with various kinds of tumor-derived MUC1 glycoproteins as well as the glycoprotein biomarker associated with interstitial pneumonia. Further, glycosylation of threonine/serine residues outside the epitope strongly influenced the antibody/epitope interaction.
Two glycoforms of the tetrapeptide GSTA (SEQ ID NO:2) with GalNAcα1-O-Ser/Thr (Tn) and NeuAcα2-6GalNAcα1-O-Ser/Thr (sialyl-Tn or STn) O-glycosylation have also been reported as a cancer-specific immunodominant epitope found in the VNTR. Tarp, M. A., et al. Glycobiology 2007; 17(2):197-209. Vaccines based on this glycopeptide were capable of overriding tolerance in human MUC1 transgenic mice and induced humoral immunity. Sorensen, A. L., et al. Glycobiology 2006; 16:96-107.
In addition, autoantibodies directed to MUC1 have been reported in healthy persons and in patients with lung cancer; however, the levels in lung cancer patients were significantly lower than those in normal individuals. Hirasawa, Y, et al. Am. J. Respir. Crit. Care Med. 2000; 161:589-594. These authors also reported that the one-year survival rate of patients with higher concentrations of autoantibody was significantly higher than that of patients with lower levels of autoantibody and suggested that the degree of decrease in autoantibody level may be associated with a patient's prognosis. Autoantibodies in breast cancer patients that are indicators of the outcome of disease have also been reported by von Mensdorff-Pouilly, S., et al. J. Clin. Oncology 2000; 18(3):574-583. In this study, detection of the antibodies in pretreatment sera had significant prognostic value and was associated with significant benefit in survival, suggesting that vaccination of patients with MUC1-derived (glyco)peptides may influence the outcome of disease. However, in this study, the authors reported that the levels of antibody were significantly higher in the breast cancer group and the benign breast tumor group than in healthy women.
Autoantibodies to aberrant O-glycopeptide epitopes are therefore of interest as biomarkers for detection of cancer. Based on a failure in one study to detect IgG autoantibodies to peptide epitopes in human sera, it has been suggested that autoantibody biomarker discovery strategies should be directed to the cancer-associated aberrant posttranslational modifications of MUC1. Such “O-glycopeptide combination epitopes” include both the peptide backbone and the cancer-associated posttranslational modification hapten structure. Wandall, H., et al. Cancer Res 2010; 70(4):1306-13. Although the aberrantly glycosylated O-glycopeptidome of MUC1 has great potential as a source of targets for cancer-associated autoantibodies, there are thousands of distinct MUC1-derived O-glycoproteins that may be combined with numerous distinct aberrant O-glycan structures, any of which may or may not prove to be useful targets for autoantibody-based detection of cancer. Such discovery is further complicated by the presence in sera of natural antibodies to cancer-associated truncated carbohydrate haptens. There continues to be a need for discovery of additional MUC1-derived O-glycopeptide combination epitopes and larger glycopeptides containing such epitopes for use as therapeutic cancer vaccines and for generating specific therapeutic antibodies. Such O-glycopeptide combination epitopes and antibodies targeting them are also desirable for use in diagnostic and prognostic assays for evaluation of the presence or risk of cancer. The invention disclosed herein meets these needs.